The present application is the U.S. national phase under 35 U.S.C xc2xa7317 of International Application No. PCT/EP98/00127, filed Feb. 23, 1998.
The present invention relates to a new use of Interleukin-15 (IL-15). The invention further relates to pharmaceutical preparations, containing IL-15 itself, IL-15 stimulating compounds or IL-15 inhibiting and/or eliminating compounds.
The cytokine interleukin-15 (IL-15) was originally identified in culture supernatants of the simian kidney epithelial cell line CV-1/EBNA and the T cell leukemia cell line HuT-102 (Grabstein et al., 1994; Burton et al., 1994; Bamford et al., 1994). The IL-15 cDNA sequence encodes a 162 amino acid (aa) precursor protein consisting of a 48 aa peptide and a 114 aa mature protein (Grabstein et al., 1994). Although there is no sequence homology with IL-2, analysis of the amino acid sequence predicts that IL-15, like IL-2, is a member of the four xcex1 helix bundle cytokine family. Furthermore IL-15 and IL-2 exert their biological activities through binding on the IL-2Rxcex2 and xcex3chains, supplemented by a specific IL-15Rxcex1 and IL-2Rxcex1 polypeptide, respectively (Giri et al., 1995). This sharing of receptor subunits probably accounts for the similar functional activities of both cytokines observed on T, B and NK cells. IL-15 mRNA is widely distributed in fibroblasts, epithelial cells and monocytes but not in resting or activated T cells, the predominant source of IL-2.
IL-15 and IL-2 share various biological functions. IL-15, like IL-2, has been defined as a T cell growth factor. IL-15 was originally discovered as a factor that could induce proliferation of the IL-2 dependent murine cytotoxic T-cell line (CD8+) CTLL-2 (Grabstein et al., 1994). Proliferation upon addition with IL-15 was also observed in phytohaemagglutinin (PHA)-activated CD4+ or CD8+ human peripheral blood T lymphocytes (PBT), and xcex3xcex4 subsets of T cells (Grabstein et al., 1994; Nishimura et al., 1996). Studies with phenotypically memory (CD45RO+) and naive (CD45ROxe2x88x92) T cells, isolated from human PET, revealed that IL-15, like IL-2, induces in memory CD4+ and CD8+ T cells and naive CD8+ T cells but not in naive CD4+ T cells the expression of the CD69 activation marker and proliferation (Kanegane et al., 1996). IL-15 was as effective as IL-2 in the in vitro generation of alloantigen-specific cytotoxic T cells in mixed lymphocyte cultures and in promoting the induction of lymphokine activated killer (LAK) cells (Grabstein et al., 1994). Additionally, in vivo studies in a murine model demonstrated the capacity of IL-15 to augment CD8+ T-cell-mediated immunity against Toxoplasma gondii infection (Khan and Kasper, 1996). Here vaccination of mice with soluble parasite antigen (Ag) and IL-15 resulted in significant proliferation of splenocytes expressing the CD8+ phenotype and protection against a lethal parasite challenge for at least 1 month postimmunization.
Natural Killer (NK) cells are considered an important target for IL-15 action. Treatment of NK cells with IL-15 results in proliferation and enhancement of cytotoxic activity and in production of Interferon xcex3 (IFNxcex3), tumor necrosis factor xcex1 (TNFxcex1) and granulocyt-macrophage colony stimulating factor (GM-CSF) (Carson et al., 1994). Furthermore IL-15 can substitute for the bone marrow microenvironment during the maturation of murine NK1.1+ cells from nonlytic to lytic effector cells (Puzanov et al., 1996).
Apart from its activities on T and NK cells, IL-15 costimulates, in a comparable way as IL-2, proliferation of B cells activated with immobilized anti-IgM or phorbol ester (Armitage et al., 1995). Stimulation of B cells with a combination of CD40L and IL-15 efficiently induces immunoglobulin synthesis. IL-15 has no stimulatory activity on resting B cells.
IL-15 was also found to have other biological activities. Chemoattractant factors are cytokines or chemokines that regulate the migration of lymphocytes to inflammation regions.
IL-15 is described as a chemoattractant factor for human PBT, inducing polarisation, invasion of collagen gels and redistribution of adhesion receptors (Wilkinson and Liew, 1995; Nieto et al., 1996). Murine mast cells proliferate in response to IL-15, but not to IL-2, using a novel receptor/signalling pathway, not shared with IL-2 (Tagaya et al., 1996). Furthermore, it has been shown that IL-15 and IL-2 have different effects on differentiation of bipotential T/NK progenitor cells, with IL-15 predominantly promoting the development of TCRxcex3xcex4 T cells and NK cells (Leclercq et al., 1996). The most striking difference, however, between IL-15 and IL-2 lies in their expression patterns. The presence of IL-15 mRNA in a variety of non-lymfoid tissues indicates that the secretion of the cytokine is not solely regulated by the immune system and/or that the cytokine can act outside the immune system itself. Accordingly, addition of IL-15 to a myoblast cell line affects parameters associated with skeletal muscle fiber hypertrophy, suggesting IL-15 has anabolic activities and increases skeletal muscle mass (Quinn et al., 1995).
Activated CD4+ T lymphocytes play a key role in the development of an effective immune response against pathogens by providing the growth factors necessary for the expansion of the activated CD4+ T lymphocytes (autocrine growth) and for the expansion of CD8+ cytolytic cells and the differentiation of B cells into antibody-secreting plasma cells (paracrine xe2x80x9chelperxe2x80x9d activity).
After clearance of the pathogen, a subfraction of the generated Ag-specific T cells persist as memory cells, either in the lymphoid tissue or in the circulation. Throughout this application, xe2x80x9cmemory cellsxe2x80x9d are defined as antigen-experienced cells. These memory lymphocytes are small, resting cells which are optimally primed for the generation of a quantitatively and qualitatively superior, secondary response upon a re-encounter with the priming Ag. In order to accomplish the transition from activated CD4+ effector cell to resting CD4+ memory cell and to acquire long-term survival, these effectors need to acquire the following characteristics:
(i) being resistant towards, or escaping from, activation-induced cell death (AICD); AICD is responsible for attenuation of the immune reaction;
(ii) being independent from autocrine growth factors, produced during the immune response. Normally, the disappearance of these growth factorsxe2x80x94a consequence of the ending of immune activityxe2x80x94results in growth factor depletion-induced cell death by apoptosis;
(iii) having the capacity, in case of a renewed contact with the antigen, to expand maximally by production of the necessary autocrine- and paracrine-acting helper cytokines such as IL-2.
The research that resulted in the present invention, indicated that IL-15 promotes the generation and persistence of CD4+ memory cells, by promoting antigen activated CD4+ T-lymphocytes to acquire the characteristics, mentioned above:resistance towards AICD, insensitivity towards apoptosis following growth factor withdrawal at the end of the antigen stimulus and high responsiveness towards renewed antigen challenge. Resistance towards AICD and insensitivity towards apoptosis determine the survival of the CD4+T lymphocytes. Responsiveness is characterised by cell division, expansion of the cell number and production of helper cytokines.
Thus, treatment of antigen stimulated CD+ cells with IL-15, even at very low concentrations, turns off the program of cell death running in the absence of growth factor. Unlike with IL-2, survival of CD4+ T cells with IL-15 is not accompanied by DNA synthesis nor proliferation, demonstrating that IL-15 induces a resting phenotype in these cells. Moreover, the sensitivity towards AICD of CD4+ T lymphocytes, cultured in presence of IL-2, is reversed by IL-15. Restimulation of these IL-15 treated, resting T cells with a suitable antigen (Ag) presented by Ag presenting cells (APC) results in maximal cell expansion, driven by a renewed production of helper cytokines. This cell expansion is not attenuated by a massive cell death as a consequence of AICD. In contrast to what is observed for cells cultured in presence of IL-2, the above-mentioned activities of IL-15 provide a method to achieve survival of immuno competent CD4+ T lymphocytes, herewith strongly improving the secondary restimulation of CD4+ T lymphocytes. In other words, the formation of immunological CD4+ memory cells can be controlled in a positive sense, by an increased IL-15 activity, or in a negative sense, by a decreased IL-15 activity.
A first aspect of the present invention thus relates to the use of IL-15 in the manufacturing of a pharmaceutical preparation for the stimulation of the formation of memory cells. Such a stimulation can be used in a number of applications. It can be applied before, during or after vaccination to increase the efficiency of the vaccination against infection or diseases of which the pathological evolution derives, at least in part, from an inadequate CD4+ T cell-dependent immune response. Thus, diseases, where the existence of sufficient numbers of Ag-specific CD4+ memory cells is necessary to control (re-emergent) pathogens, are suitable indications for IL-15 treatment. Important but non-limiting examples of such pathogenic conditions are bacterial, parasitical or viral infections (e.g. HIV) and cancer.
Other possible indications of this approach are individuals showing hyporesponsiveness towards pathogens or vaccins, or suffering from a chronic infection or from a generally weakened immune condition. As we assume that the action of IL-15 becomes even more important towards the end of an acute immune response, promoting the subsequent quiescent period, therapeutic doses of IL-15 should preferentially be administered when the immune response is subsiding, in this way favouring the establishment and long-term survival of CD4+ memory cells.
A second aspect of the present invention relates to those cases where an unwanted or harmful CD4+ T cell-dependent immune response is (co)-responsible for disease. As an example, several reports demonstrated the involvement of autopathogenic CD4+ T cells in autoimmune conditions. As a consequence it is anticipated that blocking the activity of IL-15 will suppress the long-term survival of autoreactive CD4+ effector T cell clones as well as promote the regression of already formed autoreactive CD4+ T cells, thus resulting in beneficial effects for patients suffering from an auto-immune condition. Therapy aiming at the inhibition of IL-15 activities can be accomplished by administration of agents interfering with the binding of IL-15 to its receptor such as antagonistic anti-IL-15 antibodies or anti-IL-15Rxcex1 antibodies or the Fab or F(abxe2x80x2)2 fragments of these Ab, soluble IL-15Rxcex1, fusion proteins consisting of soluble IL-15Rxcex1 and Fc fragment, or peptides binding with high affinity on the IL-15Rxcex1 without inducing signalling. A different approach consists of inhibiting IL-15 synthesis by administration of IL-15 antisense oligonucleotides through direct vaccination of patients with naked DNA, or by gene therapy approaches.
A third embodiment of the invention further relates to a pharmaceutical preparation promoting the formation of memory cells, which preparation contains IL-15 or IL-15 promoting compounds, possibly in presence of a suitable excipient. A fourth embodiment of the invention relates to a pharmaceutical preparation inhibiting the formation of memory cells, which preparation contains IL-15 inhibiting and/or eliminating compounds, such as IL-15 antibodies or compounds that interfere with the binding of IL-15 with its receptor, such as soluble IL15Rxcex1, possibly in presence of a suitable excipient.
For the use of IL-15 according to the present invention, IL-15 can be administered by bolus injection, continuous infusion, sustained release from implants or other suitable technique. Administration may be by intravenous injection, subcutaneous injection, or parenteral or intraperitoneal infusion. IL-15 therapeutic agent will be administered in the form of a pharmaceutical composition comprising purified polypeptide in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to patients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining a mammalian IL-15 polypeptide or derivative thereof with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrans, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate diluents. Elevated levels of IL-15 can also be obtained by adoptive transfer of cells ex vivo transfected with constructs consisting of an IL-15 cDNA sequence driven by a potent promoter, or by introduction into the target cells of an IL-15 cDNA sequence after a suitable promoter.
The meaning of therapeutic in the present application is not limited to the treatment of an existing disease or condition, but comprises the use of IL-15 as support during vaccination and other profylactive treatments, where the formation of immunological memory cells is essential or helpful.